Quantifying steady-state transport of water at the cellular level with NMR

Introduction: Many MRI methods derive contrast from water interacting with the tissue microenvironment under steady-state conditions. While the physics of diffusion is well-understood, the biology of steady-state water homeostasis is not. This is particularly evident in the brain, where most of what is known about water homeostasis comes from studying the flux of water or tracers. These studies help us understand how cells maintain and regulate cell volume but tell us nothing about steady-state transmembrane water transport. Here we test whether steady-state water transport is independently regulated from cell volume by utilizing a novel measurement of water exchange in combination with diffusion NMR and Intrinsic Optical Signal (IOS) microscopy measurements of microsctructural changes. More generally, this study advances our understanding of diffusion and exchange MRI contrast mechanisms and their utility as potential biomarkers.

Materials and

Methods: We have developed real-time “hydrophysiology" methods combining NMR and optical microscopy to quantify steady-state water transport (self-diffusion and transmembrane exchange) and simultaneously characterize cell structure and function. The methods were optimized to study viable ex vivo neonatal mouse spinal cords as a model of central nervous system (CNS) tissue. The NMR methods utilize a low-field, high-gradient, single-sided permanent magnet below the sample and a solenoid RF coil around the sample. Modulation of the strong (15.3 T/m) static gradient (SG) with RF pulses can probe water diffusion on sub-micron length scales commensurate with neural cell dimensions. Novel real-time methods based on diffusion exchange spectroscopy (DEXSY) were developed for measuring exchange rates faster than 100 1/s. A wide-field inverted microscope imaged IOS from above through a gap in the RF coil during NMR recording to provide additional information about structural changes.

Results, Conclusions, and Discussions: Measured exchange rate constants were 140±16 1/s under normal conditions. Sodium–potassium pump inhibition by either ouabain (Figure 1A) or oxygen and glucose deprivation lowered the exchange rate to 36 1/s, whereas less severe perturbations such as glutamate release by activation of AMPA receptors had less of an effect (Figure 1B), suggesting exchange rates may be related to the homeostatic state. However, exchange rate is expected to scale linearly with the membrane surface-to-volume ratio such that these effects on exchange could simply be due to cell swelling rather than changes in transmembrane water transport rates.
To investigate this phenomenon further, IOS microscopy measurements, which are sensitive to cell swelling, were acquired at the same time as apparent diffusion coefficient (ADC) and exchange rate measurements during perturbations to media ion concentrations and osmolarity. ADC and IOS appeared correlated, confirming the expected link between ADC and tissue microstructure. Exchange rates did not correlate with ADC or IOS when cells were still able to maintain homeostasis during osmotic perturbations (Figure 2A), but did appear correlated after high KCl perturbations caused cells to lose homeostasis (Figure 2B). This suggests that cells can independently regulate their volume and steady-state transmembrane water transport rates. It further shows the utility of combined diffusion and exchange measurements as biomarkers of physiological homeostasis and the pathological loss of homeostasis.

Acknowledgements (Optional): NW, RR, TC, and PB were funded by the NICHD IRF, NIH. JR was funded by the NIGMS PRAT program, NIH.